The formation of bone is a dynamic process that starts during embryogenesis and continues, though remodeling, during adult life. Occasionally, bone can also be regenerated when bone repair is needed. A complex series of events, involving cellular growth and differentiation together with extracellular matrix formation, are required for bone formation. A similar sequence of events takes place during bone repair.
The process of bone repair and regeneration resembles the process of wound healing in other tissues. In general, in response to injury, mesenchymal cells from the surrounding tissue migrate into the wound site and differentiate into cartilage or bone cells. A typical sequence of events includes: hemorrhage; clot formation; dissolution of the clot with concurrent removal of damaged tissues; ingrowth of granulation tissue; formation of cartilage; capillary ingrowth and cartilage turnover; rapid bone formation (callus tissue); and, finally, remodeling of the callus into cortical and trabecular bone. Bone repair, therefore, is a complex process that involves many cell types and regulatory molecules. The diverse cell populations involved in fracture repair include stem cells, macrophages, fibroblasts, vascular cells, osteoblasts, chondroblasts, and osteoclasts.
Many growth factors are also involved in the regeneration process. These include, for example, members of the bone morphogenic protein (BMP) family, fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and members of the insulin growth factor (IGF) family. PDGF, for example, has been shown to stimulate bone cell replication and DNA synthesis both in intact calvaria and isolated rat osteoblasts. Other growth factors or hormones that have been reported to have the capacity to stimulate new bone formation include acidic fibroblast growth factor, estrogen, macrophage colony stimulating factor, and calcium regulatory agents such as parathyroid hormone (PTH).
Other regulatory factors involved in bone repair are known to include systemic hormones, cytokines, growth factors, and other molecules that regulate growth and differentiation. Various osteoinductive agents have been purified and shown to be polypeptide growth-factor-like molecules. A rich source of osteogenic growth factors is found in platelet-rich plasma. The platelets possess granules that contain such growth factors as PDGF, TGF-β and others, which aid in accelerating angiogenesis and osteogenesis.
The techniques of bone reconstruction, such as is used to reconstruct defects occurring as a result of trauma, cancer surgery or errors in development, would be improved by new methods to promote bone repair. Reconstructive methods currently employed, such as using autologous bone grafts, or bone grafts with attached soft tissue and blood vessels, are associated with significant drawbacks of both cost and difficulty. For example, harvesting a useful amount of autologous bone is not easily achieved, and even autologous grafts often become infected or suffer from resorption.
Prior methods of inducing bone growth have used synthetic implants, or matrices, to support bone growth using materials, such as collagen. In designing a bioactive matrix, particular consideration must be given to the following features: biocompatability, scaffolding (the ability of a matrix to allow migration and proliferation of tissue specific cells), filling (the capacity of filling and therefore preserving the original shape of the regeneration site), barrier effect (the ability of excluding non-related cells from repopulation of the regeneration site), and carrier function (the ability of the engineered graft to carry and deliver bioactive factors). However, one of the most important limitations in designing a bioactive matrix remains the inability to determine which of the growth factors and cell adhesion molecules eventually favor and control histogenesis.
Several groups have investigated the possibility of using bone stimulating proteins and polypeptides, to influence bone repair in vivo. However, there are many drawbacks associated with these type of treatment protocols, including the time and expense in purifying recombinant proteins. Also, once administered to an animal, polypeptides may be more unstable than is generally desired for a therapeutic agent, and they may be susceptible to proteolytic attack. Furthermore, the administration of recombinant proteins can initiate various inhibitory or otherwise harmful immune responses. Further limitations often are related to the inability of the carrier to deliver significant levels of the an active agent to the desired growth locus. For example, many materials have been tested for sustained release of PDGF. Poly-L-lactide (PLLA), although it is commonly used, appears to be resorbed too quickly. Modifications of PLLA have been proposed as polylactic-co-glycolic acid (PLGA) with an improved and prolonged resorption rate. However, in both cases, cell attachment can be limited. Also, these polyhydroxy acids can generate acidic degradation bioproducts at the implanted sites with undesirable tissue reaction. Recently, other modifications have been proposed such as the combination of PLLA with chitosan (a synthetic compound structurally similar to glycosaminoglycan in the extracellular matrix) to limit the tissue reaction due to the acidic compound and improve cell attachment. Also, collagen disks or methylcellulose gel have been used to deliver PDGF with limited results due to their rapid resorption rate. The anionic characteristic of hydroxyapatite crystals has been recently used to deliver cationic bioactive molecules such as PDGF. The bone tissue regenerated in this case is qualitatively altered by the presence of synthetic hydroxyapaptite, a non-resorbable compound. In each of these cases, some of the required properties of a biomatrix are essentially missing. In some instances scaffolding is given up to favor releasing, or in other cases is the tissue-filing that is given up in favor of scaffolding.
In addition to growth factor therapy, prior methods of inducing bone growth have contemplated the use of gene therapy. However, currently there are some limitations in delivering plasmid DNA in tissues other than liver and muscle. Initial efforts toward somatic gene therapy have relied on indirect means of introducing genes into tissues, called ex vivo gene therapy, e.g., target cells are removed from the body, transfected or infected with vectors carrying recombinant genes, and re-implanted into the body (“autologous cell transfer”). A variety of transfection techniques currently are available that can be used to transfer DNA into cells in vitro; including calcium phosphate-DNA precipitation, DEAE-Dextran transfection, electroporation, liposome mediated DNA transfer or transduction with recombinant viral vectors. Such ex vivo treatment protocols have been proposed to transfer DNA into a variety of different cell types including epithelial cells (U.S. Pat. No. 4,868,116; Morgan and Mulligan WO87/00201; Morgan et al., 1987, Science 237:1476-1479; Morgan and Mulligan, U.S. Pat. No. 4,980,286), endothelial cells (WO89/05345), hepatocytes (WO89/07136; Wolff et al., 1987, Proc. Natl. Acad. Sci. USA 84:3344-3348; Ledley et al., 1987 Proc. Natl. Acad. Sci. 84:5335-5339; Wilson and Mulligan, WO89/07136; Wilson et al., 1990, Proc. Natl. Acad. Sci. 87:8437-8441), fibroblasts (Palmer et al., 1987, Proc. Natl. Acad. Sci. USA 84:1055-1059; Anson et al., 1987, Mol. Biol. Med. 4:11-20; Rosenberg et al., 1988, Science 242:1575-1578; Naughton & Naughton, U.S. Pat. No. 4,963,489), lymphocytes (Anderson et al., U.S. Pat. No. 5,399,346; Blaese, R. M. et al., 1995, Science 270:475-480), and hematopoietic stem cells (Lim, B. et al. 1989, Proc. Natl. Acad. Sci. USA 86:8892-8896; and Anderson et al., U.S. Pat. No. 5,399,346).
To improve transfection efficiency in other tissues, several studies propose the coating of plasmid DNA with different combinations of lipids and polymers. For example, coating a DNA molecule with positively charged lipids favors uptake of DNA by the cells. Direct in vivo gene transfer has been attempted with formulations of DNA trapped in liposomes (Ledley et al., 1987, J. Pediatrics 110:1); or in proteoliposomes that contain viral envelope receptor proteins (Nicolau et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:1068); and DNA coupled to a polylysine-glycoprotein carrier complex. In addition, “gene guns” have been used for gene delivery into cells (Australian Patent No. 9068389). It has even been speculated that naked DNA, or DNA associated with liposomes, can be formulated in liquid carrier solutions for injection into interstitial spaces for transfer of DNA into cells (Felgner, WO90/11092).
Perhaps one of the greatest problems associated with currently devised gene therapies, whether ex vivo or in vivo, is the inability to transfer DNA efficiently into a targeted cell population and to achieve high level expression of the gene product in vivo. Viral vectors are regarded as the most efficient system, and recombinant replication-defective viral vectors have been used to transduce (i.e., infect) cells both ex vivo and in vivo. Such vectors have included retroviral, adenovirus and adeno-associated and herpes viral vectors. While highly efficient at gene transfer, the major disadvantages associated with the use of viral vectors include the inability of many viral vectors to infect non-dividing cells; problems associated with insertional mutagenesis; inflammatory reactions to the virus and potential helper virus production, and/or production and transmission of harmful virus to other human patients.
In addition to the low efficiency of most cell types to take up and express foreign DNA, many targeted cell populations are found in such low numbers in the body that the efficiency of presentation of DNA to the specific targeted cell types is even further diminished. At present, there is a need for improved methods for increasing the efficiency with which DNA is targeted to the targeted cell population.
Defects in the process of bone repair and regeneration are linked to the development of several human diseases and disorders, for example, osteoporosis and osteogenesis imperfecta. Failure of the bone repair mechanism is, of course, also associated with significant complications in clinical orthopaedic practice, for example, fibrous non-union following bone fracture, implant interface failures and large allograft failures. There, therefore, still exists a need for other matrices, and more efficient methods for making such matrices and methods of using such matrices for inducing bone growth, as well as for using such matrices in conjunction with gene therapy.